For microscopic analysis, a few cell types are thin enough to be viewed directly (e.g., algae, protozoa, blood, tissue cultures), but most tissues (e.g., kidney, liver, brain) are too thick to allow light to be transmitted through them. Thus, in order to be examined with a microscope, a specimen must be sufficiently thin to be transparent and must possess sufficient contrast to permit the resolution of structural detail. The tissues can be sliced into very thin sections provided they are first processed to prevent cell damage.
Commonly, two types of procedures are used in preparing specimens of tissue for microscopic examination. In one procedure a specimen is frozen, cut and mounted on a slide in an elapsed time of about 15 minutes. This so-called “frozen section” procedure has the advantage of enabling a rapid histological diagnosis to be made from the specimen, and it is frequently employed in situations where a diagnosis is necessary while a patient is on an operating table. However, the “frozen section” procedure possesses certain disadvantages in that the prepared slide does not possess the uniformity of quality of slides prepared by other methods. Thus, frozen sections are difficult to interpret, and are more likely to be misinterpreted by a pathologist than are usual permanent slides. Further, the process of freezing tissue introduces considerable artifacts in it that can make certain conditions, such as some cancers, impossible to diagnose. Thus, when the frozen section procedure is used in emergency situations, it is customary for another portion of the tissue specimen to be processed using standard fixation/paraffin embedding techniques to have tissue available for additional sections if further examination becomes necessary.
In the other procedures, a slide of relatively high quality is produced when a section of the specimen is chemically fixed and mounted in a block of paraffin. However, using conventional procedures and solutions, the time required to process a specimen of tissue for mounting in paraffin is on the order of many hours to days as compared with the minutes required to process a specimen by the frozen section procedure.
In the preparation of paraffin slides, a specimen of tissue is immersed initially in a fixing agent. The fixed specimen is then immersed in a dehydrating agent, and afterward the specimen is immersed in a clearing agent. Finally, the cleared specimen is immersed in a bath of paraffin which impregnates the specimen and permits it to be sliced into thin sections for subsequent mounting onto slides. Because of the length of time required to prepare specimens by this process, it is customary for hospital laboratories to begin processing the specimens late in the afternoon after surgeons have obtained specimens from their patients. The processing continues through the night, and slides of the specimens are available for microscopic examination the next morning. Although the slides produced according to this procedure are of higher quality than those produced by the frozen section technique, the length of time required to process specimens is too great to enable this procedure to be used in situations where time is of the essence.
Using current procedures, if time or enzyme function is critical frozen sections are the preferred process. If subcellular detail is important, other procedures must be used. Selection of the correct procedure depends on what the cell biologist is looking for and to a point, becomes an art form.
What is needed in the art is a method of fixing tissue which combines the time advantage of the frozen section with the consistency and quality of the traditional paraffin embedded tissue.